Identification of a novel RHCE*Ce (829G > A) allele associated with absence of C and e antigens expression.

BACKGROUND: The Rh blood group antigens are encoded by the RHD and RHCE genes, which possess a remarkable degree of polymorphism owing to their high homologous structures. These variants of the RH genes can lead to absence or weak expression of antigens. METHODS: Analysis of RHCE genotyping by Polymerase Chain Reaction (PCR-SSP) method specific to detect c.48G, c.48C, 109 bp insertion of IVS2, c.201A and c.307C and RhCE phenotyping, were conducted in 316 Chinese patients in previous study. One patient with discrepancy typing result was collected for further RhCE serologic typing using microcolumn gel method and tube method in saline using monoclonal antibodies. PacBio sequencing was performed for RHCE, RHD and RHAG complete sequence analysis. 3D molecular models of the protein with the wild-type and mutant residue were generated using the DynaMut web server. The effect of the mutation on the protein function was predicted by PolyPhen-2 software. RESULTS: One male patient of Chinese Han was detected with RHCE*C allele showed by PCR-SSP method but ccEE phenotype. Further PacBio sequencing identified one normal RHCE*cE allele and one RHCE*Ce allele carried a novel c.829G > A (p.Gly277Arg) variant, which the encoded amino acid located in the ninth transmembrane segment of RhCE protein. Crystallisation analysis of 3D molecular models revealed that the substitution at Arg277 leads to the formation of additional hydrogen bonds, including weak hydrogen bonds between multiple atoms. It also results in hydrophobic ion interactions between Arg277 and Ala244. This mutation is predicted to have a damaging effect on protein function. CONCLUSION: One novel RHCE*Ce allele with c.829G > A (p.Gly277Arg) variant was identified to resulting in the absence or weak expression of C and e antigens.

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

Accuracy of RHC/c genotyping in Chinese Han population.

BACKGROUND AND OBJECTIVES: The RHCE gene plays an important role in the complex and polymorphic Rh blood group system. RHCE genotyping holds significant clinical and transfusion-related implications. The objective of this study was to evaluate the accuracy of RHC/c genotyping in the Chinese Han population. MATERIALS AND METHODS: Blood samples were obtained from 653 Chinese Han blood donors. The serological RhD and RhCcEe types were determined using monoclonal antibodies. Subsequently, multiplex real-time polymerase chain reaction (PCR) analysis was performed for RHC and RHc genotyping. Additionally, exon 2 of RHCE and exon 1 of RHD were sequenced. RESULTS: The analysis in this study found 443 RhD-positive donors and 210 RhD-negative donors. Among the 653 total donors, discrepancies between the RHC genotyping results and the serological results were found in 37 individuals. Specifically, 6 false-positive RhC results in RhD-positive donors and 28 false-positive RhC results in RhD-negative donors were identified based on c.48C in RHCE exon 1. Additionally, 3 false-negative RhC results were observed in the RhD-positive donors due to a 109 bp insertion in RHCE intron 2. RHc typing demonstrated complete consistency between the real-time PCR and the serological results. CONCLUSION: In the Chinese Han population, RHC genotyping was reliable when consistent results were achieved by both c.48C-based and 109 bp insertion-based genotyping. Moreover, RHc genotyping based on c.203A and c.307C polymorphic loci demonstrated dependable performance.

Recurrent pregnancy loss in a patient with anti-Rh17.

BACKGROUND: Rh is one of the most important blood group systems in transfusion medicine. The two homologous genes RHD and RHCE are located on chromosome 1p36.11 and encode for RhD and RhCE proteins, respectively. Complex genetic polymorphisms result in a variety of antigenic expression of D, C, E, c, and e. Here, we describe a case of a young female with D-- who developed anti-Rh17 secondary to blood transfusion and had signs of haemolytic disease of the fetus and fetal death in five consecutive pregnancies. CASE DESCRIPTION: EDTA-whole blood samples were collected from the patient, husband and eight siblings for blood grouping, phenotyping, and red cell antibody screening. Extracted DNA was genotyped by SNP-microarray and massively parallel sequencing (MPS) with targeted blood group exome sequencing. Copy number variation analysis was performed to identify structural variants in the RHD and RHCE. Routine phenotyping showed all family members were D+. The patient's red blood cells were C-E-c-e-, Rh17- and Rh46- and had anti-Rh17 and anti-e antibodies. MPS showed the patient carried a wildtype RHD sequence and homozygous for RHCE (1)-D (2-9)-CE (10) hybrid gene predicted to express a D-- phenotype. CONCLUSIONS: Our patient had a rare D-- phenotype and confirmed to have RHCE/RHD hybrid gene with replacement of 2-9 exons of RHCE by RHD sequences. Unfortunately, our patient developed anti-Rh17 and anti-e antibodies due to blood transfusion and suffered fetal demise in her very first pregnancy. The adverse outcomes could have been prevented by active prenatal management.

Polymorphisms in the promoter regions of RHD and RHCE genes in the Chinese Han population.

BACKGROUND AND OBJECTIVES: The Rh blood group system is the most polymorphic human blood group system. Previous studies have investigated variants in the RHD and RHCE promoter. The relevance of these variants to the Chinese Han population is further clarified in this study. MATERIALS AND METHODS: In total, 317 donors (223 Rh D-positive [D+], including 20 Del and 94 Rh D-negative [D-]) were randomly selected. The promoter regions and exon 1 of RHD and RHCE were amplified through polymerase chain reaction (PCR) whose products were directly sequenced using forward and reverse primers. RESULTS: Expected PCR products of the RHD promoter and exon 1 were amplified in 223 D+ individuals, including 20 Del individuals, and were absent in 81 of 94 D- individuals. Expected PCR products of RHCE were observed in all donors. Two single nucleotide variants (SNVs) were observed in the RHD promoter region. Moreover, 11 SNVs were observed in the promoter and exon 1 of RHCE. rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, rs2072931 and rs586178 with strong linkage disequilibria were significantly different between the D+ and D- groups. [A;C] was the most common haplotype in the RHD promoter (NC_000001.11:g.[-1033A>G;-831C>T]). [G;T;T;A;T;A;C;G;A;C;G] was the most predominant haplotype in both total and D- groups. In D+ individuals, [A;C;T;G;C;G;C;G;C;C;C] was the most frequent haplotype in the RHCE promoter (NC_000001.11:g.[-1080A>G;-958C>T;-390T>C;-378G>A;-369C>T;-296G>A;-144C>G;-132G>A;-122C>A;28C>T;48C>G]). CONCLUSION: We speculate that the SNVs/haplotypes found in this article cannot significantly affect gene expression. The present study findings should help elucidate the molecular basis of the polymorphic expression of RHD and RHCE promoter regions.

Use of computational biology to compare the theoretical tertiary structures of the most common forms of RhCE and RhD.

BACKGROUND AND OBJECTIVES: Computational biology analyses the theoretical tertiary structure of proteins and identifies the 'topological' differences between RhD and RhCE. Our aim was to identify the theoretical structural differences between the four isoforms of RhCE and RhD using computational biological tools. MATERIALS AND METHODS: Physicochemical profile was determined by hydrophobicity and electrostatic potential analysis. Secondary and tertiary structures were generated using computational biology tools. The structures were evaluated and validated using Ramachandran algorithm, which calculates the single score, p-value and root mean square deviation (RMSD). Structures were overlaid on local refinement of 'RhAG-RhCE-ANK' (PBDID 7uzq) and RhAG to compare their spatial distribution within the membrane. RESULTS: All proteins differed in surface area and electrostatic distance due to variations in hydrophobicity and electrostatic potential. The RMSD between RhD and RhCE was 0.46 ± 0.04 Å, and the comparison within RhCE was 0.57 ± 0.08 Å. The percentage of amino acids in the hydrophobic thickness was 50.24% for RhD while for RhCE it ranged between 73.08% and 76.68%. The RHAG hydrophobic thickness was 34.2 Å, and RhCE's hydrophobic thickness was 33.83 Å. We suggest that the C/c antigens differ exofacially at loops L1 and L2. For the E/e antigens, the difference lies in L6. By contrast, L4 is the same for all proteins except Rhce. CONCLUSION: The physicochemical properties of Rh proteins made them different, although their genes are homologous. Using computational biology, we model structures with sufficient precision, similar to those obtained experimentally. An amino acid variation alters the folding of the tertiary structure and the interactions with other proteins, modifying the electrostatic environment, the spatial conformations and therefore the antigenic recognition.

RHCE and Kell genotyping and alloimmunization profile in patients with sickle cell disease in the Federal District of Brazil.

INTRODUCTION: Sickle cell disease (SCD) is the most important hemoglobinopathy worldwide. The treatment often requires phenotype-matched red blood cell (RBC) transfusions, but alloimmunization to non-ABO antigens may occur in a part of the SCD patients. The genotyping has been used for RBC antigen prediction, reducing the possibility of the alloimmunization. OBJECTIVE AND METHOD: In this study we performed the genotyping for the Kell and RHCE blood groups in samples from 77 phenotyped Brazilian SCD patients, whose alloimmunization profiles were also assessed. RESULTS: Discrepancies between genotyping and phenotyping for the RHCE and Kell blood groups systems were observed in 22.07% (17/77) of the SCD patients. We found C/c and E/e discrepancies in 11.68% and 9.09% of patients, respectively; one SCD patient (1.3%) presented a discrepancy in the Kell group. Two SCD patients with discrepancies between genotype and phenotype were alloimmunized. In total, twenty-eight patients (36.4%) developed alloantibodies, of which 55.17% were directed against antigens in the Rh system, 8.62% were directed against antigens in the Kell system and 36.20%, against other groups. Finally, the frequency of discrepancies is significantly higher in non-alloimmunized patients (30.61%), compared to alloimmunized patients (7.14%) (p = 0.0217). CONCLUSION: In part, the alloimmunization of the SCD patients may have been triggered by these discrepancies, indicating that the integration of serological and molecular tests in the immunohematology routine could help to increase the transfusion safety. However, the higher number of alloimmunized patients without discrepancies showed that reasons other than the discrepancies appear to have influenced more strongly the alloimmunization in the SCD patients in this study.

Torilis japonica Extract Suppresses the Induction of Atopic Inflammation.

As one of the major intractable allergic disorders, atopic inflammation is commonly accompanied by itching, dry skin, and inflammation. Atopic inflammation deteriorates the quality of life and has no fundamental cure, so it is crucial to urgently explore and develop natural resources for long-term treatment without any side effects. This study aimed to verify Torilis japonica extract (TJE)'s relieving effect and mechanism against atopic inflammation using skin cells and skin equivalent models, as well as to investigate torilin's effect (obtained from TJE) and other unknown components as marker compounds. Torilin concentration was verified in TJE using high-performance liquid chromatography and analyzed the unknown components using nuclear magnetic resonance spectroscopy. Furthermore, TJE's cytotoxicity, regenerative effect, and cell cycle regulation effects were confirmed using skin cells with atopic inflammation (human dermal fibroblasts and HaCaT keratinocytes) by using TNF-α and IFN-γ treatments. Consequently, TJE was demonstrated to regulate TARC and CTACK expressions as chemokines and those of interleukin-4, -5, and -13 as cytokines related to atopic inflammation. TJE was further confirmed to affect the matrix metalloproteinase-1, -2, and -9 expressions, which are essential in skin damage. Lastly, this study confirmed TJE's relieving effect against atopic inflammation through a 3D skin model and RhCE model using human dermal fibroblasts and HaCaT keratinocytes. These findings on atopic inflammation verified torilin's relieving effects and TJE's other components.

RHCE*E and RHCE*e genotype incompatibility in a southern Thai Muslim population.

CONTEXT: The formation of red cell alloantibodies resulting from both transfusion and pregnancy can cause adverse effects from allogeneic blood transfusions. Alloanti-E is commonly detected among Thai and Asian populations. AIMS: This study aimed to determine RHCE*E and RHCE*e genotype incompatibility in a southern Thai Muslim population and to compare it with those previously reported for other populations. SUBJECTS AND METHODS: Nine hundred and twenty-seven DNA samples obtained from 427 unrelated healthy blood donors from southern Thai Muslims and 500 samples from Central Thais were included. Samples were genotyped for RHCE*E and RHCE*e using an in-house polymerase chain reaction with the sequence-specific primer technique. RESULTS: Significant differences were found when we compared the allele frequencies of the RHCE*E and RHCE*e between southern Thai Muslims and Central Thais: RHCE*E 0.162 versus 0.197 and RHCE*e 0.838 versus 0.803 and also found in Chinese, American native, Japanese, Korean, Alaskan native, Hawaiian, South Asian, Brazilian Japanese-descendant, and Malay Malaysian populations (P < 0.05). In addition, the E/e incompatibilities among southern Thai Muslims and Central Thais were 24.23% and 26.71%, respectively. CONCLUSIONS: This study was the first to determine the RHCE*E and RHCE*e genotype incompatibility among southern Thai Muslims, enabling the estimation of their potential alloimmunization risk. These data could be useful to provide safe blood transfusions across ethnic populations.

Five-Years Review of RHCE Alleles Detected after Weak and/or Discrepant C Results in Southern France.

Immunohematology laboratories are regularly facing transfusion issues due to serological weaknesses. Altered (partial) RH antigens account for most of them. In some situations, RHCE variant alleles are involved. Herein we present our three-step molecular exploration, with allele frequencies, that has efficiently untangled RH2 phenotype weaknesses and discrepancies in our 2017-2021 cohort. In the last 5 years, the PACA Corse EFS molecular platform received 265 samples from healthy blood donors or patients with C and C/e typing difficulties. The first-intention technique (DNA array and real time PCR for RHCE*CeRN research) detected RHCE variant alleles in 143 cases (54%). The RHCE alleles classically found in African populations were the most frequent, with RHCE*CeRN allele in 40 cases (15%) and (C)ces haplotype type 1 and 2 in 26 cases (10%). A "CE" effect haplotype was suspected in 56 cases, due to the uncommon DCE haplotype that may explain the low C expression. When there were no RHCE*Ce or RHCE*CE alleles, we then searched for RHD polymorphisms by DNA array. We detected the RHD*DAU5 and RHD*DIVa in 18 and 7 cases respectively, suggesting that C ambiguity is related to the presence of these alleles which has never been described with DAU5. If no variant RHCE and RHD alleles were detected, we finally sequenced the 10 exons of both RHCE and RHD genes according to the clinical context and found seven new RHCE alleles. Thus, this molecular strategy would improve the knowledge of RHCE variants' expression and, thus, optimize the transfusion management.

In-house performance assessment of 3D QobuR-Reconstructed Human Cornea-Like Epithelium (RhCE) for the evaluation of eye hazard.

To replace the Draize eye irritation test (OECD Test Guideline 404), several test methods based on reconstructed cornea-like epithelium (RhCE) have been developed and adopted in the OECD TG 492. The objective of this study was to stablish the experimental procedures and evaluate the performance assessment of QobuR-RhCE, an in-house RhCE model to be used for the evaluation of eye hazard. We define the essential structural, functional and procedural elements of the test method components to help assuring that the proposed test method is based on the same concepts as the validated reference methods. Performance assessment was evaluated in accordance with the revised performance standards for the assessment of proposed similar or modified in vitro reconstructed human cornea-like epithelium and the minimum list of reference chemicals was evaluated. As result, the proposed method scored 93.3% sensibility, 60% specificity, 76.7% accuracy and 96.7% within-laboratory reproducibility (WLR), providing a similar performance in comparison to the validated reference methods. Additionally, we describe a secondary endpoint based on Transepithelial Electrical Resistance (TEER) that could be of use to better discriminate between irritants and non-irritants. Taken together the results indicate that the QobuR-RhCE test method is an accurate screening tool that can be used as a standalone alternative to evaluate ocular irritation.

Impedance-based non-invasive assay for ocular damage prediction on in vitro 3D reconstructed human corneal epithelium.

Reconstructed human cornea-like epithelium (RhCE) holds unprecedented promise for toxicological analyses and the replacement of animal use. However, current standards to evaluate potential ocular irritancy present a major downfall, the need to invasively alter tissue samples to evaluate cell viability. In this study, the applicability of impedance analysis was validated by monitoring the change in cell capacitance during tissue maturation and before and after chemical application using coupled electrodes. Our results indicate that cell maturation on RhCE models can be evaluated during model production using capacitance sensing offering a faster and simpler quality control criteria for RhCE model usability. Additionally, cell capacitance resulted to be more sensitive in detecting slight cell damages than methods based on cell metabolism, and when integrated into OECD-approved testing strategies, capacitance sensing performed as good as currently accepted methodologies displaying 66% sensitivity, 100% specificity and 83% accuracy when evaluated at 300 Hz. In summary, a quantitative analysis to predict in vivo ocular irritation based on changes in RhCE capacitance by impedance spectroscopy is suggested. This methodology represents a non-invasive and non-destructive alternative that would enable the monitoring of reversible effects or repeated dose toxicity.

RHCE variant alleles and risk of alloimmunization in Brazilians.

Variant RHCE alleles are found mainly in Afro-descendant individuals, as well as in patients with sickle cell disease (SCD). The most common variants are related to the RHCE*ce allele, which can generate partial e and c antigens. Although RHCE variant alleles have been extensively studied, defining their clinical significance is a difficult task. We evaluated the risk of RhCE alloimmunization as a consequence of partial antigens in patients with a positive phenotype transfused with red blood cell (RBC) units with the corresponding antigen. A retrospective study was performed with Brazilian patients, evaluating the number of antigen-positive transfused RBC units (incompatible due to partial antigen) in 27 patients with SCD carrying RHCE variant alleles who did not develop antibodies as well as evaluating the variants present in 12 patients with partial phenotype and correlated antibody (one patient with SCD and 11 patients with other pathologies). Two patients showed variant alleles with molecular changes that had not yet been described. Variant RHCE alleles were identified in a previous study using molecular methods. RHCE*ceVS.01 was the most frequent allele found among the patients without antibodies. Six patients with partial c antigen had a mean of 3.8 c+ RBC units transfused, and 10 patients with partial e antigen were exposed for a mean of 7.2 e+ RBC units. Among the variant alleles found in alloimmunized patients, the most frequent was RHCE*ceAR, which was found in five patients; the antibodies developed were anti-hrS and/or anti-c. Our results showed that RHCE*ceVS.01 is indeed the most frequent variant allele in our cohort of patients with SCD, but the partial antigens that were identified have low risk of alloimmunization. RHCE*ceAR is the most impactful variant in the Brazilian population with high risk of alloimmunization and clinically significant anti-hrS formation.

H2 Production by Methane Oxy-Reforming: Effect of Catalyst Pretreatment on the Properties and Activity of Rh-Ce0.5Zr0.5O2 Synthetized by Microemulsion.

Green hydrogen introduction in hard-to-abate processes is held back by the cost of substituting steam reforming plants with electrolyzers. However, green hydrogen can be integrated in properly modified reforming processes. The process proposed here involves the substitution of steam reforming with oxy-reforming, which is the coupling of the former with catalytic partial oxidation (CPO), exploiting the pure oxygen coproduced during electrolysis to feed CPO, which allows for better heat exchange thanks to its exothermic nature. With the aim of developing tailored catalysts for the oxy-reforming process, Ce0.5Zr0.5O2 was synthetized by microemulsion and impregnated with Rh. The Ce-based supports were calcined at different temperatures (750 and 900 °C) and the catalysts were reduced at 750 °C or 500 °C. Tuning the calcination temperature allowed for an increase in the support surface area, resulting in well-dispersed Rh species that provided a high reducibility for both the metal active phase and the Ce-based support. This allowed for an increase in methane conversion under different conditions of contact time and pressure and the outperformance of the other catalysts. The higher activity was related to well-dispersed Rh species interacting with the support that provided a high concentration of surface OH* on the Ce-based support and increased methane dissociation. This anticipated the occurrence and the extent of steam reforming over the catalytic bed, producing a smoother thermal profile.

RHCE diversity among Brazilian patients with sickle cell disease (SCD) and selected groups of blood donors.

BACKGROUND: Several centers have selected Black donors to prevent Rh alloimmunization of patients with sickle cell disease (SCD). As the Brazilian population is considered very admixed and race definition by self-declaration is questionable, this study aimed to compare RHCE diversity among patients with SCD and selected groups of Brazilian blood donors to define which group of donors would be the adequate red cell supply for patients with SCD. METHOD: We compared RHCE allele frequencies between patients with SCD and four groups of Brazilian blood donors: self-declared Black donors (SDB), donors with predominant African genetic markers (AAM), donors with weak D expression (WDD), and random donors (RDs). Variant RHCE alleles were identified using molecular protocols. RESULTS: Among patients with SCD, 47% had at least one variant RHCE, in SDB and WDD this frequency was higher, 53% and 58.6%, respectively. In AAM and in RD the frequencies were 32% and 27.6%, respectively. In patients with SCD and SDB, the most common alleles were RHCE*ce.01, RHCE*ceVS.01, and RHCE*ceVS.02. WDD had a high frequency of RHCE*ceAR and highest frequency of variant RHCE in both alleles, followed by patients with SCD and SDB. CONCLUSION: This study showed that even in an admixed population the selection of SDB donors is the best choice of matching for transfusion support in patients with SCD. For specific RHCE alleles, selection of donors with weak D expression could be a good option.

D--phenotype due to RHD-RHCE hybrid transcript in a case of severe haemolytic disease of newborn with anti-Rh 17(Hrₒ) antibodies.

BACKGROUND: D antigen is one among the most immunogenic antigens and is the most common cause of Haemolytic Disease of Fetus and Newborn (HDFN). The D-phenotype is a rare Rh variant in which none of the RhCE antigens are expressed on the red cell surface. Individuals having D-phenotype are capable of producing a rare alloantibody named as anti-Rh17(Hr° ) in response to pregnancy or transfusion and has the potential to react with C/c and E/e antigens causing severe haemolytic transfusion reaction (HTR) and haemolytic disease of fetus and newborn (HDFN). CASE REPORT: We have encountered a case of severe HDFN with an accidental discovery of D- phenotype of the mother with anti-Rh-17 antibodies. D- phenotype has been confirmed with molecular typing along with genotyping of all family members. CONCLUSION: Rare phenotypes like D- individuals especially if allo-immunised are of great concern at times of transfusion requirements. Hence, proper identification of these individuals are important to contribute them to the rare donor pool and to adopt adequate patient blood management strategies.

Serological screening and genetic analysis of RhCE variants in the Chinese Southern Han donors.

OBJECTIVES: To screen RhCE variants in the Chinese Southern Han donors for molecular genetic analysis. BACKGROUND: More than hundreds of RhCE variant alleles have been described to resulting in weak and/or partial expression of RhCE antigens, generation of low-prevalence antigens and/or absence of a high-prevalence antigen of Rh system, which mainly reported in the people of African origin. In this study, the serological screening and molecular genetic analysis of RhCE variants were performed in the Chinese Southern Han donors. METHODS: The blood samples of E(+) donors were preliminarily collected. Then, RhCE antigens of the E(+) samples were further typed by using two sets of monoclonal anti-C, anti-c, anti-e and another anti-E. When weak expression of RhCE antigens was found, direct sequencing for 10 exons of RHCE gene, RH genotyping analysis by using multiplex ligation-dependent probe amplification, flow cytometric analysis and even cDNA sequencing were performed. RESULTS: A total of 4487 E(+) samples were collected and four samples with weak expression of antigens were detected. RHCE*Ce375G and RHCE*Ce667T variant alleles were identified in two samples with weak expression of e antigen, respectively. But no variant alleles were found in another two samples with weak expression of C antigen. CONCLUSION: The variant RHCE*Ce375G validated by mRNA sequencing and the deduced RHCE*Ce667T alleles were firstly identified in the Chinese population. The DCE haplotype might account for the weak expression of C antigen in two donors.